Nucleic acid library generation methods and compositions

ABSTRACT

Methods of forming a library of polynucleotide templates for DNA sequencing are disclosed. The methods include distributing a nucleic acid sample into different droplets in a set of emulsion droplets. The droplets into which the polynucleotide species are distributed include primers which binds to the polynucleotide species and are extended by a polymerase contained within the droplet. A set of extended primers are created simultaneously in a large number of droplets. An enriched set of extended primers are distributed into a second set of emulsion droplets containing a second primer which is extended by a polymerase contained within the second set of droplets.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application No. 62/215,626, filed Sep. 8, 2015, the contents of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

Embodiments described herein are in the field of nucleic acid molecular biology as used in microdroplet technology.

BACKGROUND

DNA sequencing is undergoing rapid development in the past several years. Highly efficient DNA sequencing systems are commercially available. A critical step in the use of these new DNA sequencing technologies is the preparation of nucleic acid libraries for sequencing. It is of interest to provide new and improved methods of generating libraries. Many nucleic acid library techniques involve a nucleic acid amplification step or steps. Such amplification steps can introduce representational biases into the libraries. Such application biases can make it difficult to obtain an accurate analysis of the nucleic acid samples under study. Accordingly, applicants provide herein new and improved methods and compositions for generating nucleic acid libraries for use in numerous DNA sequencing systems.

SUMMARY

In one embodiment is a method of forming a library of polynucleotide templates that can be as templates for DNA sequencing. The method comprises the steps of distributing a nucleic acid sample into different droplets in a set of emulsion droplets such that the plurality of the droplets comprise a single polynucleotide species, e.g., a single polynucleotide. The droplets into which the polynucleotide species are distributed comprise a primer having a region complementary to the polynucleotide species, a region for binding to a first universal primer sequence, and a purification binding moiety, e.g. biotin. The primer may optionally comprise a unique identifier sequence (UM). The primer binds to the polynucleotide species by hybridization to the target specific sequence complementary region on the polynucleotide species. The primer is subsequently extended by a polymerase contained within the droplet (as well as the other reagents, e.g. nucleotide triphosphates, required for polymerization). Because the extension of the primers is occurring simultaneously in a large number of droplets, a set of extended primers is created. The droplets are then broken so as to release the extended primers. An enriched set of extended primers is created, by removing unwanted polynucleotide fragments present in the sample and unextended primers. Various purification reagents can be used to create the enriched set of extended primers, e.g., AMPURE™ beads. The enriched set of extended primers are distributed into a second set of emulsion droplets. This second set of emulsion droplets contain a second primer comprising a region complementary to the extended primer and a region for binding a second universal primer sequence. The primer is subsequently, extended by a polymerase contained within the droplet (as well as the other reagents, e.g. nucleotide triphosphates, required for polymerization).

In some embodiments, the extended second primer is bound to a solid support by means of the purification binding moiety present on the first extended primer. The extended second primer can then be obtained by washing the bound polynucleotide constructs so as to remove unbound polynucleotides, followed by denaturation to release the polynucleotide template comprising the extended second primer.

In some embodiments, the released polynucleotide template is distributed into a third set of emulsion droplets. The third set of emulsion droplets contains a pair of adapter primers that bind to the universal primer binding sequences added by the first and second primers. These primers can be used to introduce sequences complementary to primers used for bridge PCR or other forms of clonal amplification.

In some embodiments the first adapter primer may comprise a sample index sequence. In some embodiments the second adapter primer may comprise a sample index sequence. In some embodiments the first and second adapter primer may comprise a sample index sequence.

In some embodiments the first adapter primer may comprise a unique identifier sequence. In some embodiments the second adapter primer may comprise a unique identifier sequence. In some embodiments the first and second adapter primer may comprise a unique identifier sequence.

In some embodiments the first adapter primer may comprise a unique identifier sequence and a sample index sequence. In some embodiments the second adapter primer may comprise a unique identifier sequence and a sample index sequence. In some embodiments the first and second adapter primer may comprise a unique identifier sequence and a sample index sequence.

In some embodiments the region complementary to the polynucleotide species on the first primer may be a random sequence. In some embodiments the region complementary to the polynucleotide species on the second primer may be a random sequence. In some embodiments the region complementary to the polynucleotide species on the first primer and the second primer may be a random sequence.

The above embodiments and implementations are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they may be presented in association with a same, or a different, embodiment or implementation. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and/or implementations. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative implementations, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the above embodiment and implementations are illustrative rather than limiting.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and exemplified suitable methods and materials are described below. For example, methods may be described which comprise more than two steps. In such methods, not all steps may be required to achieve a defined goal and the invention envisions the use of isolated steps to achieve these discrete goals. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The term “nucleic acid” as used herein generally refers to molecules that include naturally occurring nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), or artificial nucleic acid analogs such as peptide nucleic acid (PNA), morpholino and locked nucleic acid, glycol nucleic acid, and threose nucleic acid. Nucleic acid molecules can be obtained from an animal, plant, bacterium, fungus, viral particles or preparations, or any other biological organism. In certain embodiments, the nucleic acid molecules isolated from a single cell, tissue comprising many cells, or from cell free samples. Nucleic acid molecules can be obtained from an organism or from a biological sample obtained from an organism, e.g., from blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. Nucleic acid molecules can also be isolated from cultured cells, such as a primary cell culture or a cell line. The cells or tissues from which template nucleic acids are obtained can be infected with a virus or other intracellular pathogen.

Generally, nucleic acid can be extracted from a biological sample by a variety of techniques such as those described by Maniatis, et at, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). Nucleic acid molecules may be single-stranded, double-stranded, or double-stranded with single-stranded regions (for example, stem- and loop-structures).

The term “distributing” as used herein refers to the process of placing polynucleotides in droplets, typically an aqueous droplet surrounded by an immiscible fluid.

The term “nucleic sample” as used herein polynucleotides for analysis, typically obtained from biological source. For example a sample genomic DNA. A nucleic sample can be substantially manipulated prior to use in the subject methods. For example a cDNA preparation can be considered a nucleic acid sample analysis.

The plurality of different polynucleotide species as used herein refers to a diverse set of polynucleotides differing in sequence from one another. For example a set of DNA fragments derived from a single genome can be a plurality of different polynucleotide species. Similarly DNA fragments from a cDNA preparation can be a plurality of different polynucleotide species.

The term “primer” as used herein refers to polynucleotides and analogs thereof that are capable of selectively hybridizing to a target nucleic acid or a “template,” a target region flanking sequence or to a corresponding primer-binding site of an amplification product; and allows detection of a double-stranded nucleic acid formed by hybridization or the synthesis of a sequence complementary to the corresponding polynucleotide template, flanking sequence or amplification product from the primer's 3′ end. Typically a primer can be between about 10 to 100 nucleotides in length and can provide a point of initiation for template-directed synthesis of a polynucleotide complementary to the template, which can take place, in the presence of appropriate enzyme(s), cofactors, substrates such as nucleotides and the like.

The term “extend” or “extending” as used herein, refers to the template-specific addition of a plurality of nucleotides to a primers

The term “index sequence” or “sample index sequence”, as used herein, refers to a polynucleotide sequence that is used to identify a specific polynucleotide sample source, e.g., a specific patient. The index sequence may be of any length, but is typically 1-25 bases in length. The preferred length of an index sequence will be a function of the number of samples that need to be identified. For example a three base sequence has 64 possible sequences and thus can be used to uniquely identify 64 samples.

The term “unique identifier sequence” or UID as used herein refers to a specific nucleic acid sequence that is used to identify a single polynucleotide molecule. Using a large number of unique identifier sequences, a correspondingly large number of single polynucleotide molecules may be identified. The use of unique identifier sequences is described, among other places, in the SAFE sequencing system described in published PCT application WO2012/142213A2 The term unique identifier sequence can include barcodes for sample indexing.

The term “solid phase reversible immobilization beads” as used herein refers to solid phase beads and may be used to selectively bind polynucleotide sequences independent of the specific sequence, but having other binding characteristic, e.g. length, double-strandedness, and the like.

The term “Purification binding moiety” refers to a member of pair of compounds that bind together with high avidity and high specificity. Exemplary of such pairs of binding members are biotin and avidin, biotin and streptavidin, antibodies and cognate antigens. Either member of a purification binding moiety may be conjugated to the polynucleotide. Typically, the smaller member of the specific binding pair is conjugated to the polynucleotide. For example in the case of a biotin-avidin pair, the biotin moiety is conjugated to the polynucleotide.

The term “random” as used herein with respect to polynucleotide sequences refers to polynucleotide regions that are not designed to hybridized to specific genetic locus (other than the sequence complementary to the random region). A random sequence may be or virtually any length, but is typically between 4 and 16 bases in length. Although the term random is employed, a random sequence may be deliberately designed to have a specific sequence.

The term “universal primer sequence” refers to a region of a polynucleotide sequence that is in common (or later be in common after subsequent manipulations) in a population of diverse polynucleotide sequences. The universal primer sequence can be used to bind primers for amplification of polynucleotide sequences located between two universal primer sequences, such universal primer sequences being different from each other or identical to each other (although not strictly identical, but having the complement one sequence being identical to the other). Amplification primers may contain universal primer binding sequences and may be used to introduce such universal primer binding sequences during extension of the primer either in PCR or other primary extension techniques.

The term “region for binding a universal primer sequence” refers to the region of a polynucleotide sequence complementary to the universal primer binding sequence.

The term “region” as used herein refers to specific portion of a polynucleotide, including synthetic oligonucleotides, and extended oligonucleotides. A specific region may have a function, for example binding to a specific polynucleotide sequence by virtue of sequence complementarity, or being a universal primer binding site. A given region has a physical location. A polynucleotide may contain one or more regions.

The term “Library” as used herein refers to a set of diverse polynucleotide species having a common vector joined to the polynucleotide species. For example a whole genome that has been fragmented and attached to a plasmid can form a genetic library, wherein each member of the genetic library has the same plasmid sequence, but a different insert sequence. Similarly DNA amplification can produce a library in which primer sequences are conserved between amplicons, but differ significantly in the regions between the common sequences introduced by the amplification primers.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings. In the drawings, like reference numerals indicate like structures, elements, or method steps and the leftmost digit of a reference numeral indicates the number of the figure in which the references element first appears (for example, to be updated). All of these conventions, however, are intended to be typical or illustrative, rather than limiting. TS is used to indicate the region complementary to the polynucleotide species, e.g. a target specific sequence. UID is used indicate a unique identifier sequence. B is used to indicate biotin, ILF is used indicate a universal primer binding sequence for forward strand synthesis. ILR is used indicate a universal primer binding sequence for reverse strand synthesis. Read1 and Read2 are primer binding sequences suitable for use in bridge PCR and sequence reads in a high throughput DNA sequencing system such as Illumina HiSEQ system. RND is a random primer sequence.

FIG. 1 shows sheared DNA that is emulsified (distributed into droplets) with a biotinylated first primer comprising a target specific sequence, a unique identifier sequence, and a universal primer binding sequence. Primer extension is allowed to proceed in a polymerase catalyzed extension reaction. The emulsion is broken and the extended DNA products (double-stranded) are purified using AMPure beads to obtain a set of extended first primers. The extended first primers are emulsified (distributed into droplets) with second primers comprising a target specific sequence and a universal primer binding site. The second primers are extended.

FIG. 2 shows a continuation of the process shown in FIG. 1. The extended second primers (including the template in which extension took place) are bound to streptavidin coated beads by means of the biotin introduced in the first primer. The unbound strand is then removed by washing. The released strand is then compartmentalized by emulsification with index adapters for bridge PCR suitable for use in an Illumina DNA sequencing system. Amplification then takes place and the library produced is sequenced in an Illumina DNA sequencing system.

FIG. 3 shows a variation of the procedure in FIGS. 1 and 2, in which random primer sequences are used rather than target specific sequences in order to generate randomly primed strand synthesis. Such methods may be used for structural variant of the genome. Such methods are useful for checking structural variants such as deletions or insertions without requiring knowledge of the preexistence of specific deletions or insertions.

FIG. 4 shows a variation of the procedure in FIGS. 1 and 2 in which nested amplification takes place. A random priming sequence is included in second strand synthesis. After the reverse strand synthesis occurs a nested, biotinylated primer is used in a third emulsion (compartmentalization) process for another extension in which nested PCR products are created for subsequent sequencing.

FIG. 5 shows a variation of the procedure in FIGS. 1 and 2 for use in whole genome or whole exome sequencing. The first and second primers used random sequences for priming. When multiple primers bind to the same strand, the primers can be displaced by the processivity of the polymerase or the exonuclease activity of the polymerase.

FIG. 6 shows a variation of the procedures depicted in FIGS. 1 and 2 in which sequences are enriched for using bait probe sequences targeted to specific polynucleotide regions of interest in the genome. After emulsion PCR, the emulsion has broken and make probes are attached to a streptavidin coated particle, bringing over the polynucleotide sequences hybridized to the bait probe. The captured sequences are then denatured and released. Random forward and reverse primers can be used to convert the captured all of those to a library for sequencing.

FIG. 7 shows an example of target specific amplification generation of the library using the method depicted in FIGS. 1 and 2 in which the gene K-ras is targeted. The figure shows that at each stage of the process digital PCR is used to confirm that copies are made as predicted.

FIG. 8 shows a feasibility test of the subject method which digital PCR is used to confirm the presence of the suspected variants in the sense and antisense strands.

DETAILED DESCRIPTION OF THE INVENTION

The analysis of individual polynucleotide molecules in large quantities is a valuable molecular biology technique. However the sequence analysis of individual polynucleotide molecules using previously available methods can result in the introduction of various biases, e.g. amplification bias that interferes with obtaining meaningful analysis of experimental results.

One or more of embodiments of the subject inventions provided herein can provide one or more advantages over previously available methods. Not all embodiments of the subject invention will necessarily such advantages, either individual advantages, or multiple advantages. Such advantages include the highly efficient amplification of single molecules, the detection of amplification errors, the ability to process a large number of single molecules, and the reduction of nucleic acid amplification biases.

Various embodiments of the subject methods employ techniques in which nucleic acid amplification, e.g., PCR, takes place in an emulsion droplet. The droplets are subsequently broken after the amplification process is performed, thereby releasing the amplified nucleic acids. The amplified polynucleotides are then subjected to one or more modifications or enrichments. The modified or enriched polynucleotides are subsequently partitioned in to emulsion droplets, where they are subjected to additional modifications within the partitions.

In some embodiments, the region complementary to the polynucleotide species in the first or second primer (including embodiments of both the first and second primer) are random sequences. Random sequences may be employed for non-targeted amplification. Non-targeted amplification is a particular use for amplifying all members of a polynucleotide population for analysis, e.g., whole genomes, cDNA libraries, total RNA populations, and the like.

The nucleic acid sample comprising a plurality of different polynucleotide species is distributed into a first set of emulsion droplets so as provide a single polynucleotide species in a droplet. In some embodiments a single polynucleotide may be present in a droplet. In other embodiments, more than one polynucleotide may be present in a droplet, however a single molecule of each species will be present in each droplet. For example, a given droplet may contain several thousand different polynucleotides, but many of the polynucleotides sequences present in the droplet will be present in only a single copy, thus providing for the ability to provide clonal amplification of a single polynucleotide species starting from a single polynucleotide starting point.

In one embodiment is a method of forming a library of polynucleotide templates that can be used as templates for DNA sequencing. The method comprises the steps of distributing a nucleic acid sample into different droplets in a set of emulsion droplets such that the plurality of the droplets comprise a single polynucleotide species, e.g., a single polynucleotide. The droplets into which the polynucleotide species are distributed comprise a primer having a region complementary to the polynucleotide species, a region for binding to a first universal primer sequence, and a purification binding moiety, e.g. biotin. The primer may optionally comprise a unique identifier sequence (UID). The primer binds to the polynucleotide species by hybridization to the target specific sequence complementary region on the polynucleotide species. The primer is subsequently extended by a polymerase contained within the droplet (as well as the other reagents, e.g. nucleotide triphosphates, required for polymerization). Because the extension of the primers is occurring simultaneously in a large number of droplets, a set of extended primers is created. The droplets are then broken so as to release the extended primers. An enriched set of extended primers is created, by removing unwanted polynucleotide fragments present in the sample and unextended primers. Various purification reagents can be used to create the enriched set of extended primers, e.g., AMPURE™ beads. The enriched set of extended primers are distributed into a second set of emulsion droplets. This second set of emulsion droplets contain a second primer comprising a region complementary to the extended primer and a region for binding a second universal primer sequence. The primer is subsequently extended by a polymerase contained within the droplet (as well as the other reagents, e.g. nucleotide triphosphates, required for polymerization).

In some embodiments, the extended second primer is bound to a solid support by means of the purification binding moiety present on the first extended primer. The extended second primer can then be obtained by washing the bound polynucleotide constructs so as to remove unbound polynucleotides, followed by denaturation to release the polynucleotide template comprising the extended second primer.

In some embodiments, the released polynucleotide template is distributed into a third set of emulsion droplets. The third set of emulsion droplets contains a pair of adapter primers that bind to the universal primer binding sequences added by the first and second primers. These primers can be used to introduce sequences complementary to primers used for bridge PCR or other forms of clonal amplification.

In some embodiments the first adapter primer may comprise a sample index sequence. In some embodiments the second adapter primer may comprise a sample index sequence. In some embodiments the first and second adapter primer may comprise a sample index sequence.

In some embodiments the first adapter primer may comprise a unique identifier sequence. In some embodiments, the second adapter primer may comprise a unique identifier sequence. In some embodiments the first and second adapter primer may comprise a unique identifier sequence.

In some embodiments the first adapter primer may comprise a unique identifier sequence and a sample index sequence. In some embodiments, the second adapter primer may comprise a unique identifier sequence and a sample index sequence. In some embodiments the first and second adapter primer may comprise a unique identifier sequence and a sample index sequence.

In some embodiments the region complementary to the polynucleotide species on the first primer may be a random sequence. In some embodiments the region complementary to the polynucleotide species on the second primer may be a random sequence. In some embodiments the region complementary to the polynucleotide species on the first primer and the second primer may be a random sequence.

Basic techniques for using making suitable emulsion droplets and for carrying out polynucleotide manipulations in the droplets can be found in, among other places, U.S. Pat. No. 9,410,151 B2, U.S. Pat. No. 9,399,797 B2, U.S. Pat. No. 9,328,344 B2, U.S. Pat. No. 9,273,308 B2, U.S. Pat. No. 9,176,031 B2, U.S. Pat. No. 8,857,462 B2, which are hereby incorporated by reference.

The above embodiments and implementations are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they may be presented in association with a same, or a different, embodiment or implementation. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and/or implementations. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative implementations, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the above embodiment and implementations are illustrative rather than limiting.

EQUIVALENTS AND EMBODIMENTS

In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims.

Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims. 

The invention claimed is:
 1. A method of forming a library of polynucleotide templates for sequencing, the method comprising the steps of: distributing a nucleic acid sample comprising a plurality of different polynucleotide species into a first set of emulsion droplets, wherein a plurality of droplets comprise a single polynucleotide species and a first primer comprising: (1) a region complementary to the polynucleotide species; (2) a region for binding to a first universal primer sequence; and (3) a purification binding moiety; extending the first primer, whereby a set of extended first primers are created; breaking the first emulsion droplets; creating an enriched set of extended first primers; distributing members of the enriched set of extended first primers into a second set of emulsion droplets, wherein a plurality of droplets comprise a single first extended primer species and a second primer comprising: (1) a region complementary to the extended primer; and (2) a region for binding to a second universal primer sequence; and extending the second primer in the droplets, whereby a library of polynucleotide templates comprising the first and second universal primer binding sequences are created.
 2. The method of claim 1, further comprising the steps of: binding the extended polynucleotide template to a solid support though the purification binding moiety; washing the bound polynucleotide constructs to remove unbound polynucleotides; and releasing a strand of the bound polynucleotide templates, whereby a set or released polynucleotide templates is produced.
 3. The method of claim 2, further comprising: distributing the set of released polynucleotide templates into third set of emulsion droplets, wherein the droplets comprise: (1) a first adapter primer comprising a region for binding to the region for binding to the first universal primer sequence; and (2) a second adapter primer comprising a region for binding to the region for binding to the second universal primer sequence.
 4. The method of claim 3, wherein the first adapter primer or the second adapter primer comprises a sample index sequence.
 5. The method of claim 1, wherein the first primer comprises a unique identifier sequence.
 6. The method of claim 1, wherein the region complementary to the polynucleotide species on the first primer is targeted to specific sequence.
 7. The method of claim 6, wherein the region complementary to the extended first primer on the second primer is targeted to specific sequence.
 8. The method of claim 1, wherein the enriched set of extended first primers is created by a process comprising the step of contacting the set of extended primers with solid phase reversible immobilization beads.
 9. The method of claim 8, wherein the solid phase reversible immobilization beads are AMPURE BEADS™.
 10. The method of claim 1, comprising the step of sequencing a plurality of the library of polynucleotide templates.
 11. The method of claim 1, wherein the region complementary to the extended first primer on the second primer is random sequence, and the step of extending the second primer produces a plurality extended second primers having different random sequences, and the extending is catalyzed by an enzyme have strand displacement activity or 5′ to 3′ exonuclease activity.
 12. The method of claim 1, wherein the nucleic acid sample has been enriched for specific target prior to distributing polynucleotide species in the first set of emulsion droplets.
 13. The method of claim 1, wherein the nucleic acid sample is a cDNA library. 